A study was conducted to establish polymorphic variation of the mitochondrial DNA encoding the cytochrome oxidase subunit 1 (CO1) gene in Aedes albopictus isolated from 2 hot spot dengue-infested areas in the Subang Jaya District, Malaysia. A phylogenetic analysis was performed with the use of sequences obtained from USJ6 and Taman Subang Mas (TSM). Comparison of the local CO1 sequences with a laboratory strain (USM), alongside reference strains derived from the GenBank database revealed low genetic variation in terms of nucleotide differences and haplotype diversity. Four methods were used to construct a phylogenetic tree and illustrate the genetic relationship of the 37 Ae. albopictus populations based on the CO1 sequences, namely neighbor-joining (NJ), maximum parsimony (MP), maximum likelihood (ML), and Bayesian method, which revealed a distinct relationship between isolates from USJ6 and TSM. Our findings provide new information regarding the genetic diversity among morphologically similar Ae. albopictus, which has not been reported to date.

Mating behavior between Aedes aegypti and Ae. albopictus, established colony strains were examined under laboratory conditions (30-cm³ screened cages) for 5 consecutive days. The effect of selected male densities (30, 20, 10) and female density (20) on the number of swarming, mating pairs, eggs produced, and inseminated females were evaluated. Male densities significantly increased swarming behavior, mating pairs, and egg production of heterospecific females, but female insemination was reduced. Aedes aegypti males mate more readily with heterospecific females than do Ae. albopictus males. The current study suggests that Ae. aegypti males were not species-specific in mating, and if released into the field as practiced in genetically modified mosquito techniques, they may mate with both Ae. aegypti and Ae. albopictus females, hence reducing populations of both species by producing infertile eggs.

Aedes albopictus is an invasive mosquito species that transmits human-disease–causing pathogens. It is a container-inhabiting species that oviposits in resource-limited habitats. To mitigate larval competition, Ae. albopictus females may choose to distribute eggs from a single gonotrophic cycle among multiple containers through skip oviposition. With the use of individual females released in indoor and outdoor caged trials, we evaluated the oviposition choices made by gravid Ae. albopictus offered larval habitats with different qualities. Our results demonstrate that Ae. albopictus performs skip oviposition and that the degree of egg distribution is related to the quality of the larval habitat. In a 4-choice arena, individual Ae. albopictus oviposited in fewer containers when presented with ovisites of high-quality larval habitat (uncrowded conditions) compared with oviposition in low-quality (crowded conditions) larval habitats. Additionally, the females selectively oviposited in high-quality habitats when offered both low- and high-quality habitats, but distributed eggs more evenly among multiple high-quality habitats. Our results have important implications for mosquito management plans that include the use of lethal ovitraps, as well as the role of this behavior in distribution of disease-causing pathogens.

Mosquitoes in Habahe County of Xinjiang Uigur Autonomous Region in China are considered a serious nuisance problem to local residents, but little is known of their role in enzootic disease. Therefore, host-seeking behavior and virus detection in mosquitoes were investigated in this study. Adult host-seeking mosquitoes were sampled using the Centers for Disease Control and Prevention (CDC) light traps operated at three locations in June through August 2008. Nine traps were used at each location at 3 different heights (1 m, 3 m, and 5 m). Seven mosquito species from 4 genera were collected by CDC light traps in different habitats. In total, 90,055 mosquitoes were captured, of which Aedes vexans was the most abundant species, comprising 88.02% of all mosquitoes collected. The second most abundant species was Anopheles messese, which comprised about 5.86%. Other species caught were Culex modestus (2.89%), Aedes caspius (1.11%), Coquillettidia richiardii (0.61%), Ae. dorsalis (1.36%), and An. hyrcanus (0.14%). About 93.5% of Ae. vexans individuals were caught in CO₂-baited CDC light traps at 1 m above the ground. The highest numbers of Cx. modestus were caught at the highest trap level, 5 m above ground. Overall, significantly more mosquitoes of all species were collected at dusk than at dawn. Based on blood-meal analyses, Ae. vexans and An. messese fed on various vertebrate hosts, whereas Cx. modestus fed on ducks only. From a total of 335 mosquito pools tested, 10 pools of Ae. vexans were found positive for alphavirus. Comparison with the gene database revealed that the alphavirus deoxyribonucleic acid fragment obtained (GenBank accession no. HM160530) was 100% homologous at the nucleotide level to chikungunya virus isolate LK (PB) chik3408, chikungunya virus isolate SGEHICHD122508, and chikungunya virus strain FD080231. The results of this study suggest that ongoing, integrated mosquito and arbovirus surveillance is necessary in this river wetland.

The mosquito fauna of Oklahoma has not been evaluated since 1965 and no report has been published concerning species associated with urban areas in the state. Mosquito collections were conducted as part of the West Nile virus (WNV) surveillance program between April and November from 2003 to 2006, using standard collection methods. A total of 74,756 adults were collected in 26 urban centers in 16 counties of Oklahoma. Altogether, 40 species were recorded during this study period, bringing the total mosquito species recorded in Oklahoma to 62 species in 9 different genera and 18 subgenera. An updated checklist of Oklahoma mosquito fauna is included with a comparison to historical records. New state records include 3 species: Aedes muelleri, Anopheles perplexens, and Culex coronator. In addition to updating the checklist, 12 species of mosquitoes were tested for WNV. Pools of Culex pipiens complex represented the highest proportion testing positive for WNV (134/766, 17.5%), followed by Cx. tarsalis (13/192, 6.8%) and Aedes albopictus (5/215, 2.3%). West Nile virus–positive mosquitoes were detected earliest in June 2005 and latest in November 2004. Infected Cx. pipiens complex testing positive for WNV were more prevalent in the eastern and central areas of Oklahoma, whereas positive Cx. tarsalis were found mainly in the western areas of the state. This distinct geographical difference needs to be monitored and followed up to ensure optimal mosquito control efforts in Oklahoma communities with mosquito control capabilities.

Monitoring insecticide resistance of Aedes albopictus is required for implementing effective dengue and chikungunya vector control in Thailand. The World Health Organization standard susceptibility test for adult mosquitoes was used to determine the baseline susceptibility of a pyrethroid-susceptible laboratory strain of Ae. albopictus to 5 different pyrethroids (deltamethrin, permethrin, bifenthrin, cypermethrin, and α-cypermethrin). Subsequently, the respective established diagnostic doses (0.026% deltamethrin, 1.024% permethrin, 0.570% bifenthrin, 0.237% cypermethrin, and 0.035% α-cypermethrin) were used to test field-collected Ae. albopictus from Rayong, Koh Chang, and Pong Nom Ron. As expected, the laboratory strain was completely susceptible to all pyrethroid insecticides at the established concentrations. Rayong mosquitoes were found to be highly susceptible to bifenthrin, cypermethrin, and α-cypermethrin. Koh Chang mosquitoes were susceptible to only deltamethrin and permethrin. Pong Nom Ron mosquitoes were resistant to all pyrethroids tested. Routine assessment of these baseline results should guide future resistance monitoring to pyrethroid insecticides in Ae. albopictus in Thailand.

Fluorescent dyes are commonly used to help visualize insecticidal droplets or to trace movement of insecticides; however, the effect these dyes have on the insecticide’s efficacy and droplet characteristics is unknown. This study evaluated the effects of mixing Uvitex OB fluorescent dye with 5 adulticides on their efficacy in a wind tunnel. Efficacy was determined via droplet size characteristics, spray flux (active ingredient [AI] deposition), and female adult Aedes aegypti mortality. Fyfanon® ULV, Anvil® 10 10, Duet™, Aqualuer® 20-20, and Zenivex® E20, diluted with corn oil, were tested with and without the dye at maximum, minimum, and half-minimum label rates. Adulticide droplet size was not affected by the addition of dye to any of the 5 pesticides tested. Mosquito mortality was strongly correlated with AI deposition for all pesticides except Duet. There was no difference among correlation coefficients of the 5 pesticides and between coefficients of any pesticide pairs, indicating that all correlations were similar. The addition of dye slightly but nonsignificantly and nonconsistently affected mortality. It was found that the source of this variability was due to large variation in mortality among different replicates of the same treatment.

Adult mosquito surveys of Saint Kitts and Nevis (SKN) were performed in the dry season (March 16–23, 2010) in Saint Kitts, and the rainy season (October 18–25, 2010) in SKN. Biogents (BG) Sentinel Traps were set with CO₂ and BG Lure in urban, rural, mangrove, and dry forest habitats. Mosquitoes were identified to species, and reverse transcription–polymerase chain reaction was performed on potential vector species for dengue virus (DENV), chikungunya virus (CHIKV), and West Nile virus (WNV). The most abundant species during both seasons in St. Kitts were Culex quinquefasciatus, Aedes taeniorhynchus, and Aedes aegypti. There were 3 new records for Saint Kitts: Aedes tortilis, Anopheles albimanus, and Culex nigripalpus. Traps were also set in Nevis. No mosquito pool tested positive for DENV, CHIKV, or WNV.

We report the overwintering of Uranotaenia unguiculata adult females in Central Europe (Czech Republic, Hungary, Austria). This finding suggests a potential mode of winter persistence of putative novel lineage of West Nile virus in the temperate regions of Europe.

The Larvasonic™ Field Arm Mobile Wetlands Unit and SD-Mini were tested for efficacy against Culex larvae, and effects on aquatic nontarget organisms (NTO). The Field Arm provided 84.61% to 100% control of caged Culex larvae out to 0.91-m distance in shallow ditches and 60.45% control of Culex larvae at 0.61-m without any effects to caged NTO. Slow ditch treatment achieved 77.35% control compared to fast treatment (20.42%), whereas 77.65% control was obtained along edges of a neglected swimming pool, compared to near the middle (23.97%). In bucket tests, the SD-Mini provided >97% control of Culex and 85.35% reduction of immature giant water bugs, which decreased slightly (83.45%) over the monitoring period, which was not significantly different from cannibalistic damselflies (62.80%), with reduction of both being significantly higher than other NTO tested. There was a small (0.37%) reduction of dragonflies (naiads), due to cannibalism. Both Larvasonic units could effectively augment conventional larvicide operations in smaller areas without causing resistance within mosquito populations or harming NTO when used properly.

We evaluated the utility of 2 types of commercially available antigens as positive controls in the Rapid Analyte Measurement Platform (RAMP®) West Nile virus (WNV) assay. Purified recombinant WNV envelope antigens and whole killed virus antigens produced positive RAMP results and either type would be useful as a positive control. Killed virus antigens provide operational and economic advantages and we recommend their use over purified recombinant antigens. We also offer practical applications for RAMP positive controls and recommendations for preparing them.

Marker dyes are often mixed with liquid insecticide formulations prior to field applications to accurately determine the characteristics and penetration of droplets into targeted habitats. We have been using FD&C Red 40 Granular DM food dye at the rate of 20 g/liter in liquid solutions of Bacillus thuringiensis israelensis (Bti) for area-wide larvicide applications against the Asian tiger mosquito, Aedes albopictus. The Bti and dye mix ratio has been recommended by pesticide manufacturers for testing under operational conditions, but no data exist on the effects of the dye itself on mosquito larvae. We tested the effects of the FD&C Red 40 food dye in laboratory bioassays against different strains of Ae. albopictus (New Jersey and Maryland) and Culex pipiens pipiens (Utah) at rates of 0.039 to 80.0 g/liter. We also conducted field application trials to measure dye concentrations up to 100 m downwind when mixed and applied according to manufacturer instructions. In laboratory bioassays, we found that mean survival in cups with dye were significantly different from the controls beginning at 10.0 g/liter for New Jersey Ae. albopictus and at 20.0 g/liter for Maryland Ae. albopictus and Utah Cx. p. pipiens. In field application trials, we recorded a maximum volume density of 1,152.8 nl/cm² and calculated the maximum concentration of dye at 9.09 × 10⁻³ g/liter. Our results showed that although we detected greater effects of dye on Ae. albopictus in New Jersey experiments than Ae. albopictus in Maryland and Cx. p. pipiens from Utah, concentrations of the dye during operational applications were at least 1,100 times below concentrations that exhibited toxic effects for either species in the laboratory, suggesting that the dye will not interfere with accuracy of field bioassays. Our results conclusively demonstrate that the addition of the FD&C Red 40 marker dye does not alter the efficacy of the pesticide formulation by skewing results, but rather provides a valuable addition to accurately determine pesticide penetration and spectrum by discriminating between intended pesticide and other potential pollutants.

Sandfly fever group viruses in the genus Phlebovirus (family Bunyaviridae) are widely distributed across the globe and are a cause of disease in military troops and indigenous peoples. We assessed the laboratory sensitivity and specificity of the Sand Fly Fever Virus Antigen Assay, a rapid dipstick assay designed to detect sandfly fever Naples virus (SFNV) and Toscana virus (TOSV) against a panel of phleboviruses. The assay detected SFNV and TOSV, as well as other phleboviruses including Aguacate, Anahanga, Arumowot, Chagres, and Punta Toro viruses. It did not detect sandfly fever Sicilian, Heartland, Rio Grande, or Rift Valley fever viruses. It did not produce false positive results in the presence of uninfected sand flies (Lutzomyia longipalpis) or Cache Valley virus, a distantly related bunyavirus. Results from this laboratory evaluation suggest that this assay may be used as a rapid field-deployable assay to detect sand flies infected with TOSV and SFNV, as well as an assortment of other phleboviruses.

Recently, the BG-Sentinel® trap (BGS) trap has been reconfigured for increased durability during harsh field conditions. We evaluated the attractiveness of this redesigned trap, BG-Sentinel 2® (BGS2), and its novel granular lure cartridge system relative to the original trap and lure. Granular lures containing different combinations of lactic acid, ammonia, hexanoic acid, and octenol were also evaluated. Lure cartridges with all components except octenol trapped significantly more Aedes albopictus than lures containing octenol. This new granular lure combination and original BG-Lure® system were paired with BGS and BGS2 traps to compare relative attractiveness of the lures and the traps. All evaluations were conducted under field conditions in a suburban neighborhood in northeastern Florida from July to October 2014. Overall, the average numbers of Ae. albopictus collected by BGS or BGS2 were similar regardless of the lure type (i.e., mesh bag versus granules) (P  =  0.56). The functionality and durability of both trap models are discussed.

We describe and compare a new innovative backpack compressed-air sprayer (JQSX-12) to a Stihl® 450 backpack mist blower and a manually operated compression sprayer for its effectiveness as an alternative operational tool for indoor residual insecticide application to control malaria in Liberia. Advantages and physical characteristics of each sprayer and their spray atomization parameters are discussed.

Richard Floyd Darsie, Jr. (1915–2014) is the 2015 American Mosquito Control Association Memorial Lecture Honoree. He was one of the greatest mosquito taxonomists of the 20th century and died peacefully on April 10, 2014, in Grove City, PA, at the age of 99 after a professional career that spanned eight decades. Dick’s broad areas of interest and training made him a versatile scientist, teacher, and researcher. His intense interest in adult and immature mosquito morphology and taxonomy, as well as mosquito distribution and bionomics, started early in his career at two early academic postings: Franklin and Marshall College (1949–54) and the University of Delaware (1954–62). Dick would take his mosquito interests with him to postings and research projects around the world: Nepal, the Philippines, Atlanta, El Salvador, Guatemala, Fort Collins, South Carolina, Argentina, and Florida. His travels and studies would make him an international expert on mosquito taxonomy. Dick’s legacy lives on in the hundreds of students from across the globe who learned mosquito identification skills from this world-renowned mosquito taxonomist. All will proudly profess that, “I learned mosquito identification from Dr. Darsie.” And that is all that is needed to prove one’s credentials in the field, learning the art from the best there is.